Isolated DNA sequence capable of serving as regulatory element in a chimeric gene which can be used for the transformation of plants.
The present invention relates to the use of a regulatory element isolated from transcribed plant genes, of new chimeric genes containing them and to their use for the transformation of plants.
Numerous phenotypic characters associated with the expression of one or more gene elements can be integrated into the genome of plants and thus confer on these transgenic plants advantageous agronomic properties. In a nonexhaustive manner, there may be mentioned: the resistance to pathogenic agents for crops, the resistance to phytotoxic plant-protection products, the production of substances of dietary or pharmacological interest. In addition to the isolation and characterization of the gene elements encoding these various characters, an appropriate expression should be ensured. This appropriate expression may be situated both at the qualitative and quantitative levels. At the qualitative level, for example the spatial level: preferential expression in a specific tissue, or temporal level: inducible expression; at the quantitative level, by the accumulated quantity of the product of expression of the gene introduced. This appropriate expression depends, for a large part, on the presence of regulatory gene elements associated with the transgenes, in particular as regards the quantitative and qualitative elements. Among the key elements ensuring this appropriate regulation, the use of single or combined homologous or heterologous promoter elements has been widely described in the scientific literature. The use of a regulatory element downstream of the transgene was used for the sole purpose of putting a boundary which makes it possible to stop the process of transcription of the transgene, without presupposition as to their role as regards the quality or the quantity of the expression of the transgene.
The present invention relates to the use of an intron 1 isolated from plant genes as a regulatory element, of new chimeric genes containing them and to their use for the transformation of plants. It relates to an isolated DNA sequence capable of serving as a regulatory element in a chimeric gene which can be used for the transformation of plants and allowing the expression of the product of translation of the chimeric gene in particular in the regions of the plant undergoing rapid growth, which comprises, in the direction of transcription of the chimeric gene, at least one intron such as the first intron (intron 1) of the noncoding 5xe2x80x2 region of a plant histone gene. It relates more particularly to the simultaneous use of the intron 1 as a regulatory element and of promoters isolated from the same plant gene. It allows the appropriate expression, both quantitative and qualitative, of the transgenes under the control of these elements for gene regulation. This appropriate expression, obtained by the use of the present invention, may relate to characters such as: the resistance to pathogenic agents for crops, the resistance to phytotoxic plant-protection products, the production of substances of dietary or pharmacological interest. In particular, it makes it possible to confer on the transgenic plants an enhanced tolerance to herbicides by a qualitative and quantitative preferential expression of the product of expression of the chimeric genes in the regions of the plant undergoing rapid growth. This specific appropriate expression of the gene for herbicide resistance is obtained by the simultaneous use of the promoter regulatory elements and of at least one intron 1 of the histone gene of the xe2x80x9cH3.3-likexe2x80x9d type as regulatory element. Such a pattern of expression can be obtained for all the characters which are of interest, as described above, with the regulatory elements used to confer an enhanced herbicide tolerance. The present invention also relates to the plant cells transformed with the aid of these genes and the transformed plants regenerated from these cells as well as the plants derived from crossings using these transformed plants.
Among the plant-protection products used for the protection of crops, the systemic products are characterized in that they are transported in the plant after application and, for some of them, accumulate in the parts undergoing rapid growth, especially the caulinary and root apices, causing, in the case of herbicides, deterioration, up to the destruction, of the sensitive plants. For some of the herbicides exhibiting this type of behaviour, the primary mode of action is known and results from inactivation of characterized enzymes involved in the biosynthesis pathways of compounds required for proper development of the target plants. The target enzymes of these products may be located in various subcellular compartments and observation of the mode of action of known products most often shows a location in the plastid compartment.
Tolerance of plants sensitive to a product belonging to this group of herbicides, and whose primary target is known, may be obtained by stable introduction, into their genome, of a gene encoding the target enzyme, of any phylogenetic origin, mutated or otherwise with respect to the characteristics of inhibition, by the herbicide, of the product of expression of this gene. Another approach comprises introducing, in a stable manner, into the genome of sensitive plants a gene of any phylogenetic origin encoding an enzyme capable of metabolizing the herbicide into a compound which is inactive and nontoxic for the development of the plant. In the latter case, it is not necessary to have characterized the target of the herbicide.
Given the mode of distribution and accumulation of products of this type in the treated plants, it is advantageous to be able to express the product of translation of these genes so as to allow their potential expression and their accumulation in the regions of the plant undergoing rapid growth where these products accumulate. Furthermore, and in the case where the target of these products is located in a cellular compartment other than the cytoplasm, it is advantageous to be able to express the product of translation of these genes in the form of a precursor containing a polypeptide sequence allowing directing of the protein conferring the tolerance into the appropriate compartment, and in particular in the plastid compartment.
By way of example illustrating this approach, there may be mentioned glyphosate, sulfosate or fosametine which are broad-spectrum systemic herbicides of the phosphonomethylglycine family. They act essentially as competitive inhibitors, in relation to PEP (phosphoenolpyruvate), of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS, EC 2.5.1.19). After their application to the plant, they are transported into the plant where they accumulate in the parts undergoing rapid growth, especially the caulinary and root apices, causing the deterioration, up to the destruction, of the sensitive plants.
EPSPS, the principal target of these products, is an enzyme of the pathway of biosynthesis of aromatic amino acids which is located in the plastid compartment. This enzyme is encoded by one or more nuclear genes and is synthesized in the form of a cytoplasmic precursor and then imported into the plastids where it accumulates in its mature form.
The tolerance of plants to glyphosate and to products of the family is obtained by the stable introduction, into their genome, of an EPSPS gene of plant or bacterial origin, mutated or otherwise with respect to the characteristics of inhibition, by glyphosate, of the product of this gene. Given the mode of action of glyphosate, it is advantageous to be able to express the product of translation of this gene so as to allow its high accumulation in the plastids and, furthermore, in the regions of the plant undergoing rapid growth where the products accumulate.
It is known, for example, from American patent 4,535,060 to confer on a plant a tolerance to a herbicide of the above type, in particular N-phosphonomethylglycine or glyphosate, by introduction, into the genome of the plants, of a gene encoding an EPSPS carrying at least one mutation making this enzyme more resistant to its competitive inhibitor (glyphosate), after location of the enzyme in the plastid compartment. These techniques require, however, to be improved for greater reliability in the use of these plants during a treatment with these products under agronomic conditions.
In the present description, xe2x80x9cplantxe2x80x9d is understood to mean any differentiated multicellular organism capable of photosynthesis and xe2x80x9cplant cellxe2x80x9d any cell derived from a plant and capable of constituting undifferentiated tissues such as calli, or differentiated tissues such as embryos or plant portions or plants or seeds. xe2x80x9cIntron 1 of Arabidopsis as a regulatory elementxe2x80x9d is understood to mean an isolated DNA sequence of variable length, situated upstream of the coding part or corresponding to the structural part of a transcribed gene. Gene for tolerance to a herbicide is understood to mean any gene, of any phylogenetic origin, encoding either the target enzyme for the herbicide, optionally having one or more mutations with respect to the characteristics of inhibition by the herbicide, or an enzyme capable of metabolizing the herbicide into a compound which is inactive and nontoxic for the plant. Zones of the plants undergoing rapid growth are understood to mean the regions which are the seat of substantial cell multiplications, in particular the apical regions.
The present invention relates to the production of transferred plants having an enhanced tolerance to herbicides accumulating in the zones of the treated plants undergoing rapid growth, by regeneration of cells transformed with the aid of new chimeric genes comprising a gene for tolerance to these products. The subject of the invention is also the production of transformed plants having an enhanced tolerance to herbicides of the phosphonomethylglycine family by regeneration of cells transformed with the aid of new chimeric genes comprising a gene for tolerance to these herbicides. The invention also relates to these new chimeric genes, as well as to transformed plants which are more tolerant because of a better tolerance in the parts of these plants undergoing rapid growth, as well as to the plants derived from crossings using these transformed plants. Its subject is also new intron 1 of a plant histone and its use as regulatory zone for the construction of the above chimeric genes.
More particularly, the subject of the invention is a chimeric gene for conferring on plants especially an enhanced tolerance to a herbicide having EPSPS as target, comprising, in the direction of transcription, a promoter element, a signal peptide sequence, a sequence encoding an enzyme for tolerance to the products of the phosphonomethylglycine family and a regulatory element, characterized in that the regulatory element comprises a fragment of an intron 1 of a plant histone gene in any orientation relative to its initial orientation in the gene from which it is derived, allowing the preferential expression and the accumulation of the protein for tolerance to the herbicide in the zones for accumulation of the said herbicide.
The histone gene, from which intron 1 according to the invention is derived, comes from a monocotyledonous plant such as for example wheat, maize or rice, or preferably from a dicotyledonous plant such as for example lucerne, sunflower, soya bean, rapeseed or preferably Arabidopsis thaliana. Preferably, a histone gene of the xe2x80x9cH3.3-likexe2x80x9d type is used.
The signal peptide sequence comprises, in the direction of transcription, at least one signal peptide sequence of a plant gene encoding a signal peptide directing transport of a polypeptide to a plastid, a portion of the sequence of the mature N-terminal part of a plant gene produced when the first signal peptide is cleaved by proteolytic enzymes, and then a second signal peptide of plant gene encoding a signal peptide directing transport of the polypeptide to a sub-compartment of the plastid. The signal peptide sequence is preferably derived from a gene for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) according to European patent application PCT 508 909. The role of this characteristic sequence is to allow the release, into the plastid compartment, of a mature polypeptide with a maximum efficiency, preferably in a native form.
The coding sequence which can be used in the chimeric gene according to the invention comes from a herbicide tolerance gene of any phylogenetic origin. This sequence may be especially that of the mutated EPSPS having a degree of tolerance to glyphosate.
The promoter element according to European patent application PCT 507 698 may be of any origin, in a single or duplicated or combined form of a gene naturally expressed in plants, that is to stay, for example of bacterial origin such as that of the nopaline synthase gene, or of viral origin such as that of the 358 transcript of the cauliflower mosaic virus, or preferably of plant origin such as that of the small subunit of the ribulose-1,5-bisphosphate carboxylase/oxygenase or preferably such as that of a plant histone gene and preferably from Arabidopsis thaliana. A histone gene of the xe2x80x9cH4xe2x80x9d type is preferably used.
The chimeric gene according to the invention may comprise, in addition to the above essential parts, an untranslated intermediate zone (linker) between the promoter zone and the coding zone as well as between the coding zone and intron 1 and which may be of any phylogenetic origin.
The following examples show by way of illustration, but with no limitation being implied, several aspects of the invention: isolation of the introns according to the invention and their use for the genetic transformation of plants as well as the improved qualities of expression of the heterologous genes of plants transformed with the aid of these introns. References to xe2x80x9cCurrent Protocols in Molecular Biologyxe2x80x9d are to Volume 1 and 2, Ausubel F. M. et al., published by Green Publishing Associate and Wiley Interscience (1989) (CPMB).